Oil sands process-affected water (OSPW) is the water contained in tailings impoundment structures in oil sands operations. There are concerns about the environmental impacts of the release of OSPW because of its toxicity. In this study, ozonation followed by biodegradation was used to remediate OSPW. The impacts of the ozone process evolution on the naphthenic acids (NAs) speciation and acute toxicity were evaluated. Ion-mobility spectrometry (IMS) was used to preliminarily separate isomeric and homologous species. The results showed limited effects of the ozone reactor size on the treatment performance in terms of contaminant removal. In terms of NAs speciation, high reactivity of NAs with higher number of carbons and rings was only observed in a region of high reactivity (i.e., utilized ozone dose lower than 50 mg/L). It was also found that nearly 0.5 mg/L total NAs was oxidized per mg/L of utilized ozone dose, at utilized ozone doses lower than 50 mg/L. IMS showed that ozonation was able to degrade NAs, oxidized NAs, and sulfur/nitrogenated NAs. Complete removal of toxicity toward Vibrio fischeri was achieved after ozonation followed by 28-day biodegradation period. In vitro and in vivo assays indicated that ozonation reduced the OSPW toxicity to mice.

Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.

Unlike mammals, bony fish have two type II interferons, IFN␥ and IFN␥rel, whose pro-inflammatory functions have not been fully characterized. To elucidate the distinct roles of these type II interferons of bony fish, we examined the effects of recombinant goldfish (rg) IFN␥ and IFN␥rel on the macrophage antimicrobial responses, immune gene expression, and their signaling pathways. Our findings indicate that rgIFN␥ and rgIFN␥rel possess unique capacities to mediate each of the above processes. Q-PCR analysis revealed similar expression of both cytokines in tissues and immune cell populations of the goldfish, although IFN␥ mRNA levels were generally higher in most tissues and cell types. Whereas rgIFN␥ had long-lasting effects on the priming of goldfish monocyte ROI production, the rgIFN␥rel had relatively short-lived ROI priming potential and eventually down-regulated the priming of ROI production induced by rgIFN␥ or rgTNF␣2. Whereas rgIFN␥ induced relatively modest phagocytic and nitric oxide responses of goldfish macrophages, rgIFN␥rel induced significantly higher phagocytosis, iNOSA and iNOSB gene expression and nitric oxide production compared with rgIFN␥. The rgIFN␥ and rgIFN␥rel induced different gene expression profiles in goldfish monocytes. These differences included significantly higher induction of TNF␣2, CXCL8, ceruloplasmin, and interferon regulatory factor (IRFs) expression after activation of monocytes with rgIFN␥rel. The rgIFN␥rel was more abundant in whole cell lysates compared with rgIFN␥. Both cytokines induced the phosphorylation of Stat1, while the nuclear localization of Stat1 was only observed following treatment of monocytes with rgIFN␥. Our findings suggest the presence of functional segregation of the induction of macrophage antimicrobial functions by type II interferons of bony fish.

Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcγRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcγRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcγRIIA and mutants that associated with lipid rafts efficiently activated NF-κB, in an ERK-dependent manner. In contrast, WT FcγRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcγRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcγRIIA-mediated responses, like NF-κB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.

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